The invention is concerned with a method for producing HIV capsids in a cell-free extract. Also described are capsid intermediate compositions, auxiliary proteins, and screening assays that measure the ability of drugs to inhibit this process.
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The protein shell of the HIV virion, termed the HIV capsid or core, is composed of approximately 1500 copies of the Pr55 Gag structural protein precursor (Gelderblom, 1991). For proper assembly of the capsid to occur, Pr55 chains must undergo myristoylation (Gheysen, et al., 1989; Gottlinger, et al., 1989), an N-terminal modification thought to occur co-translationally (Towler, et al., 1988). The myristoylated chains are targeted to the host plasma membrane where assembly takes place concomitant with RNA encapsidation. As capsids are formed, they bud into the plasma membrane. This results in envelopment and subsequent release of viral particles from the cell. Coincident with their release, the immature viral particles undergo a maturation process, involving proteolytic processing of the precursor structural proteins and condensation of the capsids into collapsed, electron-dense cores (Gelderblom, 1991; Wills and Craven, 1991).
The manner in which HIV capsids assemble differs from that of many other retroviruses. Other retroviruses of the type B and type D category assemble xe2x80x9cpreformedxe2x80x9d capsids in the cytoplasm of the infected cells. Such preformed capsids are then transported to other areas of the cell, such as the plasma membrane. In contrast, HIV capsids and other type C retroviruses form in intimate association with the plasma membrane, as described above. This important characteristic of HIV capsid formation has been demonstrated through electron microscopic studies (reviewed by Gelderblom, 1991; Wills and Craven, 1991).
Analyses of various mutants of Pr55 have revealed key domains required for efficient capsid assembly and targeting to the plasma membrane (see for example Gheysen, et al., 1989; Gottlinger, et al., 1989; Trono, et al., 1989; Royer, et al., 1991; Jowett, et al., 1992; Facke, et al., 1993; Wang and Barklis, 1993; Spearman, et al., 1994; Hockley, et al., 1994; Zhao, et al., 1994). However, the actual mechanisms involved in coordinating the formation of an HIV capsid from 1500 Gag monomers have not been elucidated. Many important questions about HIV capsid assembly remain unanswered, including whether assembly is an energy-dependent process, whether host proteins are required for assembly to take place, and whether assembly proceeds by way of discrete intermediates.
A major obstacle to addressing these questions experimentally has been the inherent difficulty of studying capsid assembly in cellular systems. In cells, many of the events in question proceed extremely rapidly and are not readily amenable to manipulation, making it difficult to identify trans-acting factors and energy substrates that may be required for assembly.
Development of a cell-free system that recreates capsid biogenesis would greatly facilitate a biochemical dissection and mechanistic understanding of capsid formation. Moreover, such a system would be useful as a screening assay for identifying drugs that interfere with the process.
It is the discovery of the present invention that immature HIV capsids can be assembled in a cell-free protein translation system, when certain key components are added to the reaction. Capsid formation by this method has the same requirement as capsid formation in vivo, including a requirement for myristoylation of Gag and an apparent requirement for membranes. Furthermore, in the present invention, this method for cell-free assembly of HIV capsids is used to reveal the existence of previously unknown steps in HIV virus formation. This system has now been used to demonstrate that capsid formation is dissociable into co- and post-translational phases, each of which has distinct co-factor and/or energy requirements. The reactions that occur during the post-translational phase are dependent on ATP and at least two independent host factors which are distinguished by their differential sensitivities to non-ionic detergents. This system can be used as a screening assay or selection assay for identification of new compounds that interfere with capsid formation, and hence with production of infectious virus.
Included in this invention is the discovery that formation of HIV capsids proceeds by way of a pathway of previously unrecognized assembly intermediates, in both cells and in the cell-free system. Such intermediates have utility, for example, in the design of drugs (including peptides and antibodies) and vaccines that interfere with progression from one intermediate to the next, in the design of drugs that act by inhibiting host cell machinery involved in capsid formation, and in the design of assay systems that examine the efficacy and mechanism of action of drugs that inhibit capsid formation.
In a related aspect, also forming part of the invention are host proteins that are involved in HIV capsid formation. An exemplary host protein, termed HP68, is a 68 kD protein present in a cell-free fraction of wheat germ extract and which forms part of one or more of the intermediate complexes described above. This protein is useful as a component of the cell-free translation systems and methods described above. It has further utility in the design of drugs that block or alter its association with HIV Gag and which therefore prevent formation of immature HIV capsids.
Forming yet another related aspect of the present invention, is the discovery that pieces of genomic HIV RNA can be encapsidated into the HIV capsids produced in the cell-free system by adding such RNA to the system. This feature of the invention can be used to design of drugs that interfere with encapsidation and in the design of assay systems that examine the mechanism of actions of drugs that inhibit encapsidation.
In one aspect, the present invention is concerned with a cell-free system for translation and assembly of retroviral capsids and capsid intermediates. In particular, the invention includes a cell-free system for translation/assembly of capsids and capsid intermediates of human immunodeficiency virus (HIV).
The cell-free translation system includes (i) a cell-free translation mixture, (ii) an mRNA molecule encoding a Gag Pr55 protein derived from human immunodeficiency virus (HIV), (iii) myristoyl coenzyme A, (iv) a detergent-sensitive fraction derived from eukaryotic cell membranes, and (iv) a eukaryotic cell component characterized by insensitivity to a concentration of at least 0.5% (wt/vol) xe2x80x9cNIKKOLxe2x80x9d detergent.
Generally, in this context, the cell-free translation mixture includes sufficient cellular machinery and components to support protein translationxe2x80x94transfer RNA, ribosomes, a full complement of at least 20 different amino acids and an energy source, which may be ATP and/or GTP, as discussed herein. While the cell-free translation mixture be derived from any of a number of cell types known in the art, in a preferred embodiment, the cell-free extract in the mixture is a wheat germ extract. In another preferred embodiment, the system is also charged with HIV genomic RNA or a fraction thereof, in which case the system is capable of making capsids with such RNA encapsidated within. In yet another preferred embodiment, the system can be supplemented with additional or exogenous host cell protein, such as HP 68 described herein, that is involved in the assembly complex formation.
This system, in any of its embodiments, may also be used to prepare capsid intermediates from HIV mutants that are defective in capsid assembly. Such mutants include, but are not limited to Pr46, Pr41, Gxcex94A, and D2, which are known in the art.
In still another preferred embodiment, the translation system is a coupled transcription-translation system, in which case it further includes (i) a DNA molecule which encodes HIV Gag Pr55, (ii) an RNA polymerase for synthesizing said mRNA, and (iii) sufficient concentrations of nucleotides ATP, UTP, GTP, and CTP to support the mRNA synthesis.
In a related embodiment, the invention includes a method of producing an HIV capsid intermediate in a cell-free system. According to this embodiment of the invention, the translation system described above, in any of its embodiments, charged with an mRNA molecule encoding an HIV Pr55 Gag protein is incubated for a period of time sufficient to assemble Gag Pr55 mRNA translation products into an immature HIV capsid.
In another related embodiment, the invention includes an isolated HIV capsid intermediate selected from a group of HIV capsid intermediates having buoyant densities selected from the group of about 10 S, about 80 S, about 150 S and about 500 S, as described herein. Such intermediates have utility, for example as components in screening assays for compounds which interfere with HIV capsid assembly. Such intermediates may include host cell proteins, such as HP 68 or homologs thereof, as described herein.
In still another related embodiment, the invention includes a method of selecting a compound capable of altering HIV capsid assembly, i.e., a screening method. The method includes adding a test compound to a reaction mixture which includes the following components: (i) a cell-free translation mixture that includes a cellular extract, tRNA, ribosomes, amino acids and an energy source, (ii) an mRNA molecule encoding HIV Gag Pr55, (iii) myristoyl coenzyme A. Preferably, the myristoyl coenzyme A is present at a concentration greater than about 0.1 micromolar in the assay. The amount of capsid assembly is then measured compared in the absence and presence of test compound. A compound is selected as a compound capable of altering HIV capsid assembly if this comparison yields a significant differences between the measured amounts of either capsid or capsid intermediate(s) formed.
In still another related embodiment, the invention includes a protein that is identified as being involved in capsid assembly, as evidenced by its association with capsid intermediates, especially intermediates B, C, and D, as described herein. Generally the protein is characterized as having a peptide region having the sequence presented as SEQ ID NO: 2, specific immunoreactivity with monoclonal antibody 23c, and an apparent molecular weight of about 68 kilodaltons. Preferably, the protein is characterized by at least 75% amino acid sequence identity to HP 68. In an exemplified embodiment, it is derived from wheat germ extract, and is identified as HP 68. It is appreciated, however, that such a protein can derived from any of a number of host cell sources, including, but not limited to human cells. The present invention teaches how to identify such other, homologous proteins.
In still a further related embodiment, the invention includes a method of inhibiting HIV capsid formation in a cell. The method includes adding to the cell a compound that has been selected for its ability to inhibit capsid formation or formation of capsid intermediate(s) in any embodiment of the cell-free translation system described herein. As a related feature, the invention also extends to provide a method of selecting compounds effective to alter HIV capsid formation in cells. According to this feature of the invention, the test compound is added to cells that are forming HIV retroviral capsids. The quantity and nature of capsid intermediates formed is measured and compared to capsids formed in control cells. The compound is selected if if the quantity or nature of intermediates measured in the presence of the compound is significantly different than the those formed in the absence of the compound. Association of host assembly protein HP 68 with capsid intermediates can be used as a measurement in such a selection method, as well.
In still a further related embodiment, the invention includes a method of encapsidating genomic HIV RNA or fragments thereof. Here, any of the embodiments of the cell-free translation system described herein are used. Genomic HIV RNA or RNA fragment is added to such a system, and is encapsidated during the reaction process.